Compositions and methods for cardiac tissue repair

ABSTRACT

The invention features compositions featuring (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1; and methods of using such compositions to reduce cardiac tissue damage associated with an ischemic event or to enhance engraftment of a cell in a cardiac tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/858,154, filed Apr. 24, 2020, which is a continuation of U.S.application Ser. No. 15/623,025, filed Jun. 14, 2017, which is now U.S.Pat. No. 10,675,329, issued on Jun. 9, 2020, which is a continuation ofU.S. application Ser. No. 14/771,747, filed Aug. 31, 2015, which is nowU.S. Pat. No. 9,707,271, issued on Jul. 18, 2017, which is the U.S.National Stage application, pursuant to 35 U.S.C. § 371, of PCTInternational Application No. PCT/US2014/022094, filed Mar. 7, 2014,designating the United States and published in English, which claimspriority to and the benefit of U.S. Provisional Application No.61/775,285, filed Mar. 8, 2013, all of which are incorporated herein byreference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grant from the NationalInstitutes of Health, Grant No: HL085210. The government has certainrights in the invention.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted electronically in XML format following conversion from theoriginally filed TXT format.

The content of the electronic XML Sequence Listing, (Date of creation:Jan. 12, 2023; Size: 9,838 bytes; Name:167914-010306USCON-Sequence_Listing.xml), and the original TXT format,is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Despite recent advances in treating ischemic injuries, stroke andmyocardial infarction continue to kill or disable vast numbers of peopleeach year. In the U.S. alone, 600,000 new myocardial infarctions and320,000 recurrent attacks occur annually. About 38 percent of the peoplewho experience a myocardial infarction in a given year will die, whilemany of those who survive will experience some loss in cardiac function.

Certain cell types, including muscle cells and neurons are particularlyvulnerable to ischemic injury in connection with myocardial infarctionand stroke. Technologies associated with the identification, isolation,and culture of stem/progenitor cells now provides many candidate cellsfor cell replacement applications in regenerative medicine. Notably,however, transplantation of culture-expanded adult stem/progenitor cellsoften results in poor cellular engraftment, survival, and migration intosites of tissue injury. As such, current cell replacement strategies fortreating myocardial infarction involving the injection ofstem/progenitor cells result in modest improvements in cardiac function,at best. Low levels of engraftment, survival, and cell replacement afterinjection of adult or embryonic stem cells into the injured leftventricle wall are important issues that reduce the potentialeffectiveness of cell replacement strategies after myocardialinfarction. Moreover, intravenous infusion of cultured adultstem/progenitor cells can be accompanied by microembolism and cardiacarrhythmias. Accordingly, improved methods of treating tissue injury,particularly ischemic injuries associated with myocardial infarction,are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods for treating or preventing cardiac tissue damage, includingdamage associated with an ischemic event.

In one aspect, the invention provides a composition containing one ormore of connective tissue growth factor (CTGF) and human C-terminal CTGFpeptide; and one or more of insulin and IGF-1.

In another aspect, the invention provides a composition containing ahuman C-terminal CTGF peptide that enhances cardiac progenitor cellsurvival and/or proliferation.

In still another aspect, the invention provides a method for increasingcardiac cell survival or proliferation, the method involving contactinga cardiac cell at risk of cell death with a composition containing oneor more of connective tissue growth factor (CTGF) and human C-terminalCTGF peptide; and one or more of insulin and IGF-1.

In yet another aspect, the invention provides a method for stabilizingor reducing cardiac tissue damage in a subject, the method involvingcontacting a cardiac cell of the subject with a composition containingone or more of connective tissue growth factor (CTGF) and humanC-terminal CTGF peptide; and one or more of insulin and IGF-1, therebystabilizing or reducing cardiac tissue damage in the subject.

In still another aspect, the invention provides a method for enhancingengraftment of a cardiac cell or progenitor thereof (e.g., an adultcardiac myocyte, adult cardiac endothelial cell, adult cardiac smoothmuscle cell, adult cardiac fibroblast, adult cardiac stem cell, adultcardiac progenitor cell, adult vascular stem cell, adult epicardialcell, adult sub-epicardial cell, adult bone marrow-derived stem orprogenitor cell, cardiac derivative from embryonic stem (ES) cells,and/or induced pluripotent stem (iPS) cell), the method involvingcontacting the cell (i.e., to be primed) with a composition containingone or more of connective tissue growth factor (CTGF) and humanC-terminal CTGF peptide; and one or more of insulin and IGF-1, therebyenhancing engraftment.

In another aspect, the invention provides a method for enhancingengraftment of a cardiac cell or progenitor thereof in a subject, themethod involving: contacting the cardiac cell or progenitor in vitrowith a composition containing one or more of connective tissue growthfactor (CTGF) and human C-terminal CTGF peptide; and one or more ofinsulin and IGF-1, thereby generating a primed cell; and administeringthe primed cell to the subject, thereby enhancing engraftment of thecell in the subject.

In one aspect, the invention provides a cellular composition containingan isolated cardiac progenitor cell, cardiac stem cell, mesenchymal stemcell or progeny cell thereof contacted with a composition containing oneor more of connective tissue growth factor (CTGF) and human C-terminalCTGF peptide; and one or more of insulin and IGF-1.

In another aspect, the invention provides an isolated polynucleotideencoding CTGF-D4.

In still another aspect, the invention provides an isolated polypeptidecomprising CTGF-D4.

In yet another aspect, the invention provides an isolated cardiacprogenitor cell, cardiac stem cell, mesenchymal stem cell or progenycell thereof expressing recombinant CTGF-D4. In certain embodiments, theisolated cardiac progenitor cell, cardiac stem cell, mesenchymal stemcell or progeny cell also expresses insulin and/or IGF-1.

In various embodiments of any of the aspects delineated herein, thecardiac cell or progenitor is contacted in vitro or in vivo. In variousembodiments of any of the aspects delineated herein, the cardiac cell orprogenitor is contacted with the composition or primed cell for 5, 10,15, 20, 25, 30, 35, 40, 45, 60, 90, 120, 150, 180, 210, 240 min, or 3,4, 5, or 6 hrs. In various embodiments of any of the aspects delineatedherein, the cardiac cell or progenitor is autologous or heterologous. Invarious embodiments of any of the aspects delineated herein, followingcontact of the cardiac cell or progenitor in vitro, the cell isadministered to a subject.

In various embodiments of any of the aspects delineated herein, thecomposition or primed cell is administered to a subject directly to asite of cardiac tissue damage or cardiac disease or is administeredsystemically. In particular embodiments, the composition or primed cellis administered by intra-arterial infusion.

In various embodiments of any of the aspects delineated herein, themethod reduces cell death or increases cardiac function. In particularembodiments, the method increases cardiac cell number or reduces cardiaccell death. In various embodiments of any of the aspects delineatedherein, the method increases cardiac cell number by at least about 5%compared to a corresponding untreated control cardiac tissue or heart.

In various embodiments of any of the aspects delineated herein, thesubject has a disease or disorder selected from the group consisting ofmyocardial infarction, congestive heart failure, stroke, and ischemia.In various embodiments of any of the aspects delineated herein, themethod prevents or ameliorates ischemic damage. In various embodimentsof any of the aspects delineated herein, the method reduces apoptosis orincreases cell proliferation. In various embodiments of any of theaspects delineated herein, the method prevents or ameliorates ischemicdamage in a cardiac tissue post-myocardial infarction.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

Definitions

By “increasing epicardial cell proliferation” is meant increasing celldivision of an epicardial progenitor cell or a cell derived from anepicardial progenitor cell in vivo or in vitro. Increasing epicardialcell proliferation may also include promoting, supporting, or inducingthe differentiation and/or migration of epicardial cells. For example,an increase in cell number may be at least about a 5%, 10%, 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% increase in the numberof epicardial cells relative to the number of cells present in anaturally-occurring, corresponding cardiac tissue or heart.

By “cardiac protective activity” is meant any biological activity thatmaintains or increases the survival or function of a cardiac cell orcardiac tissue in vitro or in vivo.

By “cardiac function” is meant the biological function of cardiac tissueor heart (e.g., contractile function). Methods for measuring thebiological function of the heart are standard in the art (e.g., Textbookof Medical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co.,2000) and are also described herein. By “increasing in cardiac function”is meant an increase in a biological function of the heart by at leastabout 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%relative to the biological function present in a naturally-occurring,corresponding cardiac tissue or heart.

By “cell survival” is meant cell viability.

By “reducing cell death” is meant reducing the propensity or probabilitythat a cell will die. Cell death can be apoptotic, necrotic, or by anyother means.

By “cellular factor” is meant any biological agent produced by a cell.While cellular factors isolated from culture media are typicallysecreted by cells in culture, the scope of the invention is intended toinclude any factor released from a cultured cell into growth media. Inone embodiment, a cellular factor of the invention is secreted by a cellor is released into culture media when a cell breaks open and releasesits contents into the growth media. Exemplary cellular factors includeconnective tissue growth factor (CTGF), human C-terminal CTGF peptide,insulin and IGF-1.

By “secreted cellular factor” is meant any biologically active agentthat a cell secretes during in vitro culture.

By “connective tissue growth factor (CTGF) polypeptide” is meant aprotein or fragment thereof having at least about 85% identity to NCBIAccession No. NP_001892, or that binds an antibody generated against theCTGF antigen. An exemplary full-length CTGF polypeptide is providedbelow.

(SEQ ID NO: 1)   1mtaasmgpvr vafvvllalc srpavgqncs gpcrcpdepa prcpagvslv ldgcgccrvc  61akqlgelcte rdpcdphkgl fcdfgspanr kigvctakdg apcifggtvy rsgesfqssc 121kyqctcldga vgcmplcsmd vrlpspdcpf prrvklpgkc ceewvcdepk dqtvvgpala 181ayrledtfgp dptmirancl vqttewsacs ktcgmgistr vtndnascrl ekqsrlcmvr 241pceadleeni kkgkkcirtp kiskpikfel sgctsmktyr akfcgvctdg rcctphrttt 301lpvefkcpdg evmkknmmfi ktcachyncp gdndifesly yrkmygdma

By “CTGF nucleic acid molecule” is meant a polynucleotide encoding aCTGF polypeptide.

In one preferred embodiment, a CTGF polypeptide is CTGF module 4(CTGF-D4), which is provided below.

(SEQ ID NO: 2)  1mgkkcirtpk iskpikfels gctsmktyra kfcgvctdgr cctphrtttl pvefkcpdge 61vmkknmmfik tcachyncpg dndifeslyy rkmygdma

In another preferred embodiment, a CTGF polypeptide is CTGF-D4 thatincludes a signal peptide (bold), which is provided below.

(SEQ ID NO: 3)   1maaasmgpvr vafvvllalc srpavgqnsc gpcrcpdepk kcirtpkisk pikfelsgct  61smktyrakfc gvctdgrcct phrtttlpve fkcpdgevmk knmmfiktca chyncpgdnd 121ifeslyyrkm ygdma

An exemplary CTGF nucleic acid molecule encoding the above CTGF-D4polypeptide and signal peptide sequence (bold) is provided below.

(SEQ ID NO: 4)   1 atggccgccg ccagtatggg ccccgtccgc gtcgccttcg tggtcctcct cgccctctgc  61agccggccgg ccgtcggcca gaactgcagc gggccgtgcc ggtgcccgga cgagccaaaa 121aagtgcatcc gtactcccaa aatctccaag cctatcaagt ttgagctttc tggctgcacc 181agcatgaaga cataccgagc taaattctgt ggagtatgta ccgacggccg atgctgcacc 241ccccacagaa ccaccaccct gccggtggag ttcaagtgcc ctgacggcga ggtcatgaag 301aagaacatga tgttcatcaa gacctgtgcc tgccattaca actgtcccgg agacaatgac 361atctttgaat cgctgtacta caggaagatg tacggagaca tggcatga

By “insulin polypeptide” is meant a protein or fragment thereof havingat least about 85% identity to NCBI Accession No. NP_000198, or thatbinds an antibody generated against the insulin antigen. An exemplaryfull-length insulin polypeptide is provided below.

(SEQ ID NO: 5)  1malwmrllpl lallalwgpd paaafvnqhl cgshlvealy lvcgergffy tpktrreaed 61lqvgqvelgg gpgagslqpl alegslqkrg iveqcctsic slyqlenycn

In one preferred embodiment, the insulin is a dimer of insulin A-chainand insulin B-chain of the full-length insulin polypeptide (A-chain:amino acids 90-110; B-chain: amino acids 25-54) linked by 2 disulfidebonds. Active or mature insulin is processed from the full-lengthinsulin polypeptide, also termed preproinsulin. The insulin signalpeptide (amino acids 1-24) is cleaved from preproinsulin to generateproinsulin. The proinsulin polypeptide folds in the endoplasmicreticulum, including the formation of intramolecular disulfide bonds.Cleavage of insulin C-chain (amino acids 57-87) from the foldedproinsulin polypeptide produces a dimer of the A-chain and B-chainlinked by 2 interchain disulfide bonds, which is the mature or activeform. The A-chain of the active or mature dimer contains an intrachaindisulfide bond. Active insulin is commercially available (Sigma-Aldrich19278; CAS No. 11061-68-0; MDL No. MFCD00131380). Insulin A- and B-chainsequences are provided below.

Insulin A-chain : (SEQ ID NO: 6) 1  giveqcctsi cslyqlenyc nInsulin B-chain: (SEQ ID NO: 7) 1  vnqhlcgshl vealylvcge rgffytpkt

By “insulin nucleic acid molecule” is meant a polynucleotide encoding aninsulin polypeptide.

By “Insulin-like growth factor 1 (IGF-1) polypeptide” is meant a proteinor fragment thereof having at least about 85% identity to NCBI AccessionNo. NP_000609, or that binds an antibody generated against the IGF-1antigen. An exemplary full-length CTGF polypeptide is provided below.

(SEQ ID NO: 8)   1mgkisslptq lfkccfcdfl kvkmhtmsss hlfylalcll tftssatagp etlcgaelvd  61alqfvcgdrg fyfnkptgyg sssrrapqtg ivdeccfrsc dlrrlemyca plkpaksars 121vraqrhtdmp ktqkevhlkn asrgsagnkn yrm

By “IGF-1 nucleic acid molecule” is meant a polynucleotide encoding aIGF-1 polypeptide.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10% change in expression levels,preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.”

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “deficiency of a particular cell-type” is meant fewer of a specificset of cells than are normally present in a tissue or organ not having adeficiency. For example, a deficiency is a 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or even 100% deficit in the number of cells ofa particular cell-type (e.g., cardiomyocytes, epicardial progenitorcells, embryonic stem cells, endothelial cells, endothelial precursorcells, fibroblasts, neurons, adipocytes) relative to the number of cellspresent in a naturally-occurring, corresponding tissue or organ. Methodsfor assaying cell-number are standard in the art, and are described in(Bonifacino et al., Current Protocols in Cell Biology, Loose-leaf, JohnWiley and Sons, Inc., San Francisco, Calif., 1999; Robinson et al.,Current Protocols in Cytometry Loose-leaf, John Wiley and Sons, Inc.,San Francisco, Calif., October 1997).

“Derived from” as used herein refers to the process of obtaining a cellfrom a subject, embryo, biological sample, or cell culture.

“Detect” refers to identifying the presence, absence or amount of theobject to be detected.

By “detectable label” is meant a composition that when linked to amolecule of interest renders the latter detectable, via spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radioactive isotopes, magnetic beads,metallic beads, colloidal particles, fluorescent dyes, electron-densereagents, enzymes (for example, as commonly used in an ELISA), biotin,digoxigenin, or haptens.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include any disease or injury that results in areduction in cell number or biological function, including ischemicinjury, such as stroke, myocardial infarction, or any other ischemicevent that causes tissue damage.

By “effective amount” is meant the amount of a required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a ischemic injury variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

By “engraftment” is meant the integration of an exogenous cell into atissue of a subject. In one embodiment, a primed cell (e.g., cardiacprogenitor cell) is engrafted into the heart of a subject in needthereof (e.g., post-myocardiac infarction).

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more of the entire length ofthe reference nucleic acid molecule or polypeptide. A fragment maycontain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 nucleotides or amino acids.

By “increase” is meant to alter positively by at least 5%. An alterationmay be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.When a cellular factor is “isolated” from a cultured epicardialprogenitor cell the cellular factor is typically separated from cellsand cellular debris. It need not be purified to homogeneity. In fact,the composition comprising an isolated cellular factor typicallycomprises any number of cellular factors whose presence contributes tothe biological activity (e.g., growth promoting, survival promoting, orproliferation promoting activity) of the composition. In one embodiment,a composition of the invention comprises or consists of conditionedmedia from which cells and cellular debris have been removed.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reduce” is meant to alter negatively by at least 5%. An alterationmay be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “primed cell” is meant a cell that is contacted with any one or moreof connective tissue growth factor (CTGF), human C-terminal CTGFpeptide, insulin and/or IGF-1 prior to administration to a subject(e.g., for engraftment in the subject). In various embodiments, a primedcell is contacted with CdM. In various embodiments, a primed cell iscontacted with any one or more of connective tissue growth factor(CTGF), human C-terminal CTGF peptide, insulin and/or IGF-1 for 5, 10,15, 20, 25, 30, 35, 40, 45, 60, 90, 120, 150, 180, 210, 240 min, or 3,4, 5, or 6 hrs., prior to administration for engraftment in a subject

By “repair” is meant to ameliorate damage or disease in a tissue ororgan.

By “tissue” is meant a collection of cells having a similar morphologyand function.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict proliferation of adult rat CPCs was induced by humanstromal cell CdM. FIG. 1A is a graph showing time course changes in thenumbers of CPCs treated with CdM or SFM (left panel) and phase contrastimages of CSCs, CPCs in CPC growth medium, and CPCs treated with CdMfrom MSCs, p75MSCs, or fibroblasts or SFM for 8 days (magnification,10×). CPC growth medium=CSC medium with 2% FBS. Scale bars=50 μM (rightpanel). CdM was assayed from 2 different donors for each cell type. Thecontrol cell number (48,896 cells) was regarded as 100%. Data aremean±SEM, n=3 to 7. *, P<0.0001 vs baseline; **, P<0.01 vs baseline; †,P<0.0001 vs SFM. FIG. 1B is a graph showing quantification ofBrdU-positive CPCs after immuno-cytochemistry. Data are mean±SEM, n=3,CdM from different donors were assayed for each cell type. Inset:Immunoblot for Ki67 in CPCs (molecular weight, 359 kDa: lane 1, SFM;lane 2, lx MSC CdM; lane 3, p75MSC CdM; lane 4, lx fibroblast CdM). *,P<0.05 vs SFM; **, P<0.01 vs SFM. FIG. 1C is a graph showing that MSCCdM did not grow adult rat cardiac fibroblasts. Data are mean±SEM, n=3.The control cell number (34,606 cells) was regarded as 100%. *, P<0.0001vs baseline. CdM, conditioned medium. SFM, serum-free a-MEM. GM, CPCgrowth medium (CSC medium with 2% FBS).

FIGS. 2A-2C depict proliferation of CPCs. FIG. 2A is a graph showing adose-dependent effect of 10×-concentrated CdM on CPC proliferation. CPCgrowth in 1×CdM from one MSC donor and one p75MSC donor is shown forreference. Control cell number (60,191 cells) was regarded as 100%. Dataare mean±SEM, n=3. 10×CdM from 2 different donors was assayed for eachcell type. *, P<0.0001 vs baseline; **, P<0.0001 vs day 4; t, P<0.05 vsday 8. CdM, conditioned medium. FIG. 2B is a graph showing growth ofCPCs in p75MSC CdM is dependent on signaling through STAT3 and abolishedby incubation with the specific STAT3 inhibitor, “Stattic” (10 μM). *,P<0.001 vs DMSO vehicle on day 2; **, P<0.001 vs. DMSO vehicle on day 4.FIG. 2C is a graph showing time course changes in the numbers of CPCstreated with SFM supplemented with various growth factors (EGF, bFGF,and LIF; 10 ng/ml) and in the absence of Insulin-Transferrin-Selenium.Control cell number (64,026 cells) was regarded as 100%. Data aremean±SEM, n=3. *, P<0.01 SFM and SFM+EGF+FGF vs baseline; **, P<0.001SFM, SFM+EGF+FGF, and SFM+LIF+EGF+FGF vs baseline. Data for growth in1×CdM from one MSC donor is shown for reference.

FIGS. 3A-3E depict STAT3 and Akt activation in CPCs treated with 1×CdM.FIG. 3A shows immunoblotting for phosphorylated STAT3 (p-STAT3) andtotal STAT3 (T-STAT3) in CPCs (molecular weight, 86 kDa) (left panel)and quantification of STAT3 phosphorylation levels (n=3) (right panel).Beta-actin levels indicate loading controls. The corrected values in SFMon day 1 and 2 were designated as 1, n=3. *, P<0.05 vs SFM. FIG. 3Bshows images of immunofluorescent staining for p-STAT3 and T-STAT3 inCPCs (magnification×400). Note: p-STAT3 localizes to CPC nuclei. Blueindicates DAPI nuclear staining. FIG. 3C shows immunoblotting forphosphorylated Akt (p-Akt) and total Akt (T-Akt) in CPCs (molecularweight, 62 kDa). FIG. 3D is a graph showing an inhibitory effect ofAG490 (10 μM) on CPC growth and survival in stromal cell CdM for 48 hrs.Data are mean±SEM, n=3 to 6. Control cell numbers (121,863 cells in MSCCdM, 115,342 cells in p75MSC CdM, and 118,682 cells in fibro CdM) wereregarded as 100%. *, P<0.0001 vs control. FIG. 3E is a graph showinginhibitory effects of AG490 (10 μM) and LY294002 (10 μM) on CPCsincubated with 1×CdM for 48 hrs. Data are mean±SEM, n=3 to 6. Thecontrol cell numbers (121,863 cells) were regarded as 100%. *, P<0.0001vs control; **, P<0.01 vs AG; †, P<0.05 vs LY. Con: control, DMSO. AG:AG490, Jak2/STAT3 pathway inhibitor. LY: LY294002, inhibitor of PI3K/Aktpathway. A+L: AG490+LY294002. CdM, conditioned medium. SFM, serum-freea-MEM. GM, CPC growth medium (CSC medium with 2% FBS).

FIGS. 4A-4C depict the protective effect of human stromal cell CdM onrat CPCs exposed to chronic hypoxia (1% oxygen for 48 hrs). FIG. 4A is agraph showing CPC numbers in GM, SFM and CdM after exposure to chronichypoxia. Control cell number (139,616 cells) was regarded as 100%. Dataare mean±SEM, n=3, CdM from 2 different donors was assayed for each celltype. *, P<0.05 vs SFM; **, P<0.01 vs SFM. FIG. 4B is a graph depictingSTAT3 inhibition with AG490 (10 μM) blocks CPC protection conferred byCdM during hypoxia. Control cell numbers (56,559 cells in MSC CdM,92,120 cells in p75MSC CdM, and 74,511 cells in fibro CdM) were regardedas 100%. *, P<0.0001 vs CdM. AG: AG490, Jak2/STAT3 pathway inhibitor.FIG. 4C is a graph showing survival of CPCs in p75MSC CdM and underhypoxic conditions for 48 hrs is dependent on signaling through STAT3and abolished by incubation with the STAT3-specific inhibitor, “Stattic”(10 μM). **, P<0.001 vs DMSO vehicle with 1× or 10×CdM. CdM, conditionedmedium. SFM, serum-free α-MEM. GM, CPC growth medium (CSC medium with 2%FBS).

FIGS. 5A and 5B depict differentiation of CPCs expanded in CdM. FIG. 5Adepicts images of immunofluorescent staining for α—SA, α-sarcomericactin; SMA, α-smooth muscle actin; and vWF, von Willebrand factor. Leftpanels (Baseline) show the CPCs in growth medium 3 days after plating,and the right panels show CPCs expanded in CdM for 4 days. Scalebars=100 μM. FIG. 5B depicts quantification of % positive cells forα—SA, SMA, and vWF. Data are mean±SEM, n=3. CdM, conditioned medium.

FIGS. 6A-6C depict intra-arterial infusion of p75MSC CdM significantlyreduced cardiac apoptosis/necrosis 48 hrs after MI. FIG. 6A depictsTUNEL staining of heart sections from vehicle (SFM-treated). Scalebars=100 μM. FIG. 6B depicts TUNEL staining of heart sections fromCdM-treated C57bl6 mice. At 24 hrs after LAD ligation, 200 μl of SFM or30×p75MSC CdM was slowly infused into the left ventricle lumen(intra-arterial delivery). Scale bars=100 μM. FIG. 6C depictsquantification of TUNEL⁺ cells in heart sections of animals thatreceived intra-arterial infusion of SFM or 30×p75MSC CdM 24 hrs afterMI.

FIGS. 7A and 7B depict representative images from echocardiographyshowing intra-arterial infusion of 30×p75MSC CdM 24 hrs after MIimproved cardiac function 1 week after MI.

FIGS. 8A-8F are graphs depicting that intra-arterial infusion of30×p75MSC CdM 24 hrs after MI improved cardiac function 1 week after MI.FIG. 8A is a graph showing that P75 CdM treatment significantly improvedwall motion (thickening) after MI. Echocardiography score was determinedwith a 13 segment model similar to the American Society ofEchocardiography's 16 segment model. The best possible score is a 13(full motion) and the worst possible score is a 39 (akinetic). FIG. 8Bis a graph showing that P75 CdM infusion significantly increased(preserved) the percent of fractional shortening after MI. FIG. 8C is agraph showing that P75 CdM significantly increased (preserved) anteriorwall thickness in diastole after MI. FIG. 8D is a graph showing that P75CdM treatment significantly increased (preserved) anterior wallthickness in systole after MI. FIG. 8E is a graph showing that there wasno significant difference in end diastolic diameter of the leftventricle with or without p75 CdM treatment after MI. FIG. 8F is a graphshowing that P75 CdM infusion significantly decreased the end systolicdiameter of the left ventricle after MI. *, P≤0.05; **, P≤0.01. For alldata, SFM, n=8; p75 CdM, n=6. Echocardiography was performed using aVisualSonics Vevo 770 system.

FIGS. 9A-9H show that priming of adult rat CSCs with human p75MSC CdMmarkedly improved CSC graft success 1 week after MI. At 1 day after MI,CSCs were primed in 30×CdM or vehicle (SFM) for 30 min. on ice prior toco-injection (2 sub-epicardial injections per rat, 1 per border zone).FIG. 9A is an image of the largest cell graft for control rats (n=5), at1 week after MI and injections of CSCs/SFM. Yellow autofluorescenceindicates host-derived myocytes. Scale bar=100 μM. FIGS. 9B-9D depictimages of sub-epicardial grafts from three different rats, 1 week afterMI and injections of CSCs/CdM (n=6). Scale bars=100 μM. Ki67 staining(red) in FIG. 9C shows proliferating GFP⁺ CSC derivatives insub-epicardial tissue after CSC/CdM injections. FIGS. 9D and 9D′ areimages showing that in hearts treated with CSCs/CdM, GFP⁺ cells migratedbetween apparently healthy cardiac myocytes in order to reach distantzones of necrotic myocardium with infarction. In FIG. 9D, white arrowsindicate change in CSC orientation during migration from sub-epicardiuminto myocardium after MI; compare to orientation in FIG. 9B. Scalebar=50 μM. In FIG. 9D′ dashed white line indicates edge of infarction.Scale bar=100 μM. FIG. 9E is an image showing that after CdM-priming,GFP⁺ CSC derivatives migrated into areas of necrosis with few remainingviable myocytes. Scale bar=50 μM. FIG. 9F is an image showing thatCdM-primed CSC derivatives differentiated into CD31-positive (red)vascular endothelial cells to repair blood vessels. Scale bar=100 μM.FIG. 9G is an image showing CdM-primed CSC derivatives differentiatedinto smooth muscle alpha actin-positive (SMA, red) smooth muscle cellsand myofibroblasts (arrows indicate co-localizations with GFP). Scalebar=100 μM. FIG. 9H is a graph depicting quantification of GFP⁺ cellsfrom individual tissue sections with the most engraftment in hearts thatreceived CSCs/SFM (rats 1-5) or CSCs/CdM (rats 6-11). Note: Counting wasstopped after 20,000 GFP⁺ cells for two of the CSC/CdM-treated rats, butobserved thousands of additional GFP⁺ cells.

FIG. 10 is an image showing the localization of CSC-derived GFP⁺ cells,1 week after MI and CSC/CdM injections. CSCs primed with 30×p75MSC CdMengrafted into sub-epicardial tissues after MI and migrated intospecific zones with infarction. Magnification, 200×.

FIGS. 11A-11D show that CTGF and Insulin are key factors present inp75MSC CdM that promoted the survival and proliferation of CPCs. FIG.11A is a graph showing that incubation of 10×p75 MSC CdM with antiseraspecific to human CTGF ablated its ability to protect CPCs during 48 hrsof hypoxia. Non-specific IgG (con IgG) or anti-CTGF was added toseparate aliquots of CdM (10 μg/ml, each). ***, P<0.001 vs. con IgG.FIG. 11B is a graph showing CTGF (3 ng/ml, 30 min. incubation) inducedp-STAT3 in CPCs compared with incubation in vehicle (1% BSA). Inset(representative blot): Levels of p-STAT3 after 30 min. incubation in1×CdM are shown for comparison. ***, P<0.001 vs. con IgG. FIG. 11C is agraph showing effects of Insulin on survival and growth of CPCs undernormoxic conditions. Inset (representative blot): Increasing Insulinconcentration had a dose-responsive effect on p-Akt levels in CPCs (30min. incubation). FIG. 11D is a graph showing that dual incubation ofCPCs with C-terminal domain 4 peptide (CTGF D4) and Insulin (1 ng/ml,each) had synergistic effects on CPC survival during 48 hrs of hypoxia.*, P<0.05 vs SFM; **, P<0.01 vs SFM. For FIGS. 11A-11D, n=3-5. CdM,conditioned medium. SFM, serum-free α-MEM.

FIGS. 12A-12C and 12A′-12C′ depict priming of cultured CSCs in CTGF-D4and Insulin promoted graft success after MI. FIGS. 12A and 12A′ areimages showing that few control rats (1/7) injected with CSCs primed for30 min. in vehicle (SFM with 1% BSA) had detectable GFP⁺ cells at 1 weekafter MI and sub-epicardial injections. Scale bars=50 μM in FIG. 12A and100 μM in FIG. 12A′. Note: GFP⁺ cells of control rats did not exitsub-epicardial graft site. All rats (5/5) that received CSCs primed withCTGF-D4 (3 ng/ml)/Insulin (30 ng/ml) demonstrated robust engraftment ofGFP⁺ cells at 1 week after MI (FIGS. 12B, 12B′, 12C and 12C′). FIG. 12Bdepicts sub-epicardial engraftment of GFP⁺ cells 1 week after MI in arepresentative animal that received CSCs primed with CTGF-D4/Insulin.Scale bar=50 μM. FIG. 12B′ depicts extensive migration/integration ofGFP⁺ cells 1 week after MI in a representative animal that received CSCsprimed with CTGF-D4/Insulin. White dashes indicate infarct border. Areabeyond dashes has few viable myocytes that remain from host. Scalebar=50 μM. FIG. 12C depicts engraftment of GFP⁺ cells 1 week after MI ina second representative animal that received CSCs primed withCTGF-D4/Insulin. Image demonstrates integration into area withinfarction. Scale bar=50 μM. FIG. 12C′ depicts extensivemigration/integration of GFP⁺ cells 1 week after MI in the secondrepresentative animal that received CSCs primed with CTGF-D4/Insulin.FITC channel shows extent of migration into infarct. White dashesindicate infarct border. Area beyond dashes has few viable myocytes thatremain from host. Scale bar=50 μM.

FIGS. 13A-13J show involvement of the Wnt pathway in the effects ofp75-CdM and CTGF-D4 on epicardial-derived cells. FIG. 13A depictsWestern blots for Lrp5/6 and phosphorylated GSK3β (p-GSK3β) in rat EPDClysates. EPDCs were incubated in serum-free medium (SFM) or p75-CdM for30 min. FIG. 13B consists of a graph depiciting the dose-response effectof CTGF-D4 on p-GSK3β in EPDCs (left panel) and an image of nuclearlocalization of β-catenin in EPDCs after CTGF-D4 exposure (40 ng/ml)(right panel). FIG. 13C is a graph depicting that Dkk-1 significantlyreduced protection of EPDCs by CTGF-D4 under hypoxic conditions (1%oxygen, 48 hrs). FIG. 13D is a graph depicting that anti-Lrp5/6significantly reduced protection of EPDCs by CTGF-D4 under hypoxicconditions (1% oxygen, 48 hrs). FIG. 13E is a graph depicting that anantibody that specifically binds Lrp6 significantly reduced protectionof EPDCs by CTGF-D4 under hypoxic conditions (1% oxygen, 48 hrs). FIG.1F is an image showing that EPDCs graft and migrate into the heart afterMI (vertical arrow: subepicardial injection site; horizontal arrows:migration at various time points). FIG. 1G is an image show that addingblocking antisera to Lrp6 to the CTGF-D4/Insulin priming mixsubstantially reduced the number of grafted EPDCs when hearts wereexamined at 1 week after MI (red: control cells labeled withnon-specific IgG; green: anti-Lrp6 cells (green). FIG. 1H is an imageshowing the difference in the number of control cells (non-specific IgG,red) and anti-Lrp6 cells (green) at locations distal to thesubepicardial graft site. FIG. 13I is a graph depicting removal of CTGFfrom p75-CdM by antibody pull-down FIG. 13J is a graph depictingdecreased expression of CTGF in p75-CdM by lentiviral transduction ofhuman p75MSCs with shRNA to CTGF.

FIGS. 14A-14E depict that CTGF-D4 increased adhesion of EPDCs tofibronectin and the expression of markers of differentiation andadhesion/migration. FIG. 14A is a graph depicting that CTGF-D4 increasedadhesion of EPDCs to fibronectin. FIG. 14B are Western blots showing theeffects of CTGF-D4 on markers of differentiation (vWF, SMA) andadhesion/migration (CD90, ETrB). FIG. 14C are Western blots showing thatadding Lrp6 to medium containing CTGF-D4 altered the expression ofmarkers of differentiation (vWF, SMA) and adhesion/migration (CD90,ETrB). Addition of Lrp6 neutralizing antisera to medium containingCTGF-D4 at the same dose as in (FIG. 14B) reversed EMT-likedifferentiation and instead promoted expression of Keratin, anepithelial cell marker. FIG. 14D is a graph depicting that apharmacological antagonist to ETrB reduced EPDC survival under hypoxicconditions (1% oxygen, 48 hrs). FIG. 14E is a graph depicting that apharmacological antagonist to FGFR1 reduced EPDC survival under hypoxicconditions (1% oxygen, 48 hrs).

FIGS. 15A-15D depict that epicardial-derived cell (EPDC) CdM providedvascular protection. FIG. 15A are images depicting FITC-albuminextravasation in heart with MI (left panel: sham; right panel, CdM).FIG. 15B is a graph showing that delivery of EPDC CdM into the leftventricle lumen (intra-arterial) reduced vascular leak after MI andreperfusion. FIG. 15C are Western Blots depicting that treatment withEPDC CdM at the time of reperfusion significantly reduced the level ofp-VE-Cadherin and significantly increased the levels of CD31. Thisindicates that EPDC CdM protected endothelial cells from reperfusioninjury. FIG. 15D are graphs depicting the relative intensity ofp-VE-Cadherin (upper panel) and CD31 (bottom panel) in MEM and EPDC-CdMtreated subjects. The level of p-VE-Cadherin decreased and the level ofCD31 significantly increased indicating that EPDC CdM protectedendothelial cells from reperfusion injury.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions comprising (a) one or more ofconnective tissue growth factor (CTGF) and human C-terminal CTGFpeptide; and (b) one or more of insulin and IGF-1; and methods of usingsuch compositions to reduce cardiac tissue damage associated with anischemic event or to enhance engraftment of a cell in a cardiac tissue.

The present invention is based, at least in part, on the discoveriesthat priming of cells for engraftment in p75MSC CdM or a definedcombination of CTGF-D4, insulin, and/or IGF-1 provides a useful way toboost graft success for clinical application of CSCs or culturedstem/progenitor cells derived from other tissues or sources.Accordingly, the invention provides therapeutic and prophylacticcompositions comprising agents secreted by mesenchymal stem cells (e.g.,CTGF, insulin, and IGF-1) and methods of using such compositions toreduce cardiac cell death, preserve cardiac function after an ischemicevent, and to generally prevent cardiac damage and promote cardiachealing or regeneration.

Mesenchymal Stem Cells

Paracrine activity from mesenchymal cells such as fibroblasts and otherstromal cells promotes tissue repair after injury and also regulates, inpart, stem cell niches. In the bone marrow, endothelial cells andstromal derivatives from non-hematopoietic progenitor cells (multipotentstromal cells, MSCs) support hematopoietic stem cells (HSCs) byproviding critical structural and regulatory components of thehematopoietic niche. The niche components include cellular substrate,e.g. extracellular matrix, as well as multiple growth factors,cytokines, and hormones that influence HSC self-renewal, proliferation,survival, and function. Due to their supportive roles, feeder layers ofstromal cells (e.g. MSCs or fibroblasts) are commonly used to supportthe culture of HSCs, other types of adult stem/progenitor cells, and EScells.

Human bone marrow contains a subpopulation of MSC that can be isolatedby magnetic-activated cell sorting against CD271 (p75 low-affinity nervegrowth factor receptor, p75MSCs). Human p75MSCs secrete diverse growthfactors and cytokines that promote cell survival, angiogenesis, and stemcell engraftment. In transplantation studies, co-infusion of human HSCsand p75MSCs into immunodeficient mice provided a 10-23 fold improvementin multi-lineage engraftment of bone marrow compared with co-infusion ofHSCs and typical (non-selected) human MSCs. CD271⁺ cells characteristicsof bone marrow p75MSCs are rapidly mobilized into the blood of patientswith acute MI. Without being bound to a particular theory, it washypothesized that circulating CD271⁺ cells participate in cardiacrepair/remodeling after myocardial infarction, in part through paracrineactivity. Experiments were performed to investigate the effects ofstromal cell-derived ligands on cardiac stem/progenitor cells(CSCs/CPCs). It was found that conditioned medium (CdM) from humanp75MSCs supported the proliferation and survival of adult rat CSCs/CPCs.Furthermore, priming of CSCs in p75MSC CdM for 30 min. prior totransplantation markedly improved CSC grafts after MI. By screeningp75MSC CdM for molecules that protected CPCs under hypoxic conditions,two ligands with synergistic effects on CSC survival, CTGF and Insulin,were identified. Priming of CSCs with a defined combination of humanCTGF C-terminal peptide (domain 4) and Insulin promoted graft successafter MI. Short-term priming of human CSCs with p75MSC CdM or CTGF-D4and Insulin/IGF-1 may improve graft success and cardiac regeneration inpatients with myocardial infarction.

Insulin and Insulin Growth Factor 1 (IGF-1)

Priming in IGF-1 was shown to improve the survival of cardiac cellgrafts with adult and embryonic stem cells. Cultured adult rat CPCs wereprotected by Insulin or IGF-1 during hypoxia. Insulin and Insulin-likegrowth factor 1 (IGF-1) bind to tyrosine kinase holoreceptors andpromote cell survival and proliferation by signaling through thePI3K/Akt and Ras/MAP kinase pathways. Partial functional redundancy forInsulin and IGF-1 signaling is evidenced by signaling through receptorheterodimers IR/IGF-1R and bidirectional cross-talk betweenligands/receptors. Although human p75MSC CdM did not contain detectableIGF-1, it had sufficient residual bovine Insulin to significantlyimprove CPC survival under hypoxic conditions. CdM contains Insulinbecause MSCs actively sample their environment and internalize fetalcalf serum components such as albumin, IgG, and Insulin from theirgrowth medium. Despite washing, they release some components back intothe base medium used for CdM production.

Connective Tissue Growth Factor (CTGF)

CTGF is a secreted “matricellular” protein with multiple functions inmammalian development and tissue remodeling/repair after injury,including angiogenesis and fibrosis. During pancreatic development, CTGFpromotes the proliferation of beta cell progentitors in islets. Cardiacexpression of CTGF increases significantly after MI and it is expressedby interstitial fibroblasts and cardiac myocytes. By interacting withthe extracellular matrix, integrins and several cell surface receptors(e.g. LRP-1, LRP-6, TrkA), CTGF mediates numerous cellular functionsincluding: adhesion, proliferation, migration, differentiation, andsurvival. To regulate coincident processes after injury such asangiogenesis and fibrosis, CTGF physically associates with numerousother secreted proteins including VEGFA, Slit3, von Willebrand Factor,PDGF-B, BMP-4, IGF-1, IGF-2, TGF alpha and TGF beta.

CTGF controls fibrosis in multiple tissues after injury, in part, byinteracting with TGF beta, IGF-1 or IGF-2 and promoting thedifferentiation of fibroblasts into myofibroblasts. The N-terminal (1st)and 2nd domains of CTGF interact with IGFs and TGF beta or BMP4,respectively. Notably, due to its numerous binding partners, the effectsof CTGF are context-dependent. In the presence of cellular mitogens suchas EGF, CTGF does not induce fibrosis, even when pro-fibrotic mediatorslike TGF beta or IGF-2 are present. C-terminal domain of CTGF (CTGF-D4)and Insulin were found to act in synergy to promote the proliferationand survival of cultured CPCs under hypoxic conditions. Furthermore, adefined combination of CTGF-D4 and Insulin promoted graft success withadult CSCs. Importantly, the CTGF-D4/Insulin priming method describedherein is unlikely to promote myofibroblast differentiation or fibrosisfrom transplanted CSCs as CTGF-D4 is known to promote cell adhesion andproliferation, but lacks N-terminal functions in fibrosis.

Formulations

In one embodiment, a composition of the invention comprises or consistsessentially of (a) one or more of connective tissue growth factor (CTGF)and human C-terminal CTGF peptide; and (b) one or more of insulin andIGF-1. In another embodiment, a composition of the invention comprises acell contacted with (a) one or more of connective tissue growth factor(CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulinand IGF-1.

The biologically active agents present in the conditioned media, thecells, or a combination thereof, can be conveniently provided to asubject as sterile liquid preparations, e.g., isotonic aqueoussolutions, suspensions, emulsions, dispersions, or viscous compositions,which may be buffered to a selected pH. Cells and agents of theinvention may be provided as liquid or viscous formulations. For someapplications, liquid formations are desirable because they areconvenient to administer, especially by injection. Where prolongedcontact with a tissue is desired, a viscous composition may bepreferred. Such compositions are formulated within the appropriateviscosity range. Liquid or viscous compositions can comprise carriers,which can be a solvent or dispersing medium containing, for example,water, saline, phosphate buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like) and suitablemixtures thereof.

Sterile injectable solutions are prepared by compositions comprising asecreted cellular factor isolated from cultures of stromal or epicardialprogenitor cells in the required amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionsmay be in admixture with a suitable carrier, diluent, or excipient, suchas sterile water, physiological saline, glucose, dextrose, or the like.The compositions can also be lyophilized. The compositions can containauxiliary substances such as wetting, dispersing, or emulsifying agents(e.g., methylcellulose), pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the cells or agents present in their conditioned media.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent,such as methylcellulose. Other suitable thickening agents include, forexample, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose,carbomer, and the like. The choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form). Those skilled in the art will recognize that thecomponents of the compositions should be selected to be chemicallyinert.

Compositions comprising (a) one or more of connective tissue growthfactor (CTGF) and human C-terminal CTGF peptide; and (b) one or more ofinsulin and IGF-1 or cells contacted with such agents are administeredin an amount required to achieve a therapeutic or prophylactic effect.Such an amount will vary depending on the conditions. Typically,biologically active cellular factors present will be purified andsubsequently concentrated so that the protein content of the compositionis increased by at least about 5-fold, 10-fold or 20-fold over theamount or protein originally present in the media. In other embodiments,the protein content is increased by at least about 25-fold, 30-fold,40-fold or even by 50-fold. Preferably, the composition comprises aneffective amount of (a) one or more of connective tissue growth factor(CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulinand IGF-1.

The precise determination of what would be considered an effective doseis based on factors individual to each subject, including their size,age, sex, weight, and condition of the particular subject. Dosages canbe readily ascertained by those skilled in the art from this disclosureand the knowledge in the art.

Optionally, the methods of the invention provide for the administrationof a composition of the invention to a suitable animal model to identifythe dosage of the composition(s), concentration of components thereinand timing of administering the composition(s), which elicit tissuerepair, reduce cell death, or induce another desirable biologicalresponse. Such determinations do not require undue experimentation, butare routine and can be ascertained without undue experimentation.

Methods of Delivery

Compositions comprising (a) one or more of connective tissue growthfactor (CTGF) and human C-terminal CTGF peptide; and (b) one or more ofinsulin and IGF-1 or cells contacted with such agents may be deliveredto a subject in need thereof. Modes of administration includeintramuscular, intra-cardiac, oral, rectal, topical, intraocular,buccal, intravaginal, intracisternal, intra-arterial,intracerebroventricular, intratracheal, nasal, transdermal, within/onimplants, e.g., fibers such as collagen, osmotic pumps, or parenteralroutes. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intraperitoneal, intragonadal or infusion.

The compositions can be administered via localized injection, includingcatheter administration, systemic injection, localized injection,intravenous injection, or parenteral administration. When administeringa therapeutic composition of the present invention, it will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion). Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage).Compositions of the invention can be introduced by injection, catheter,or the like. Compositions of the invention include pharmaceuticalcompositions comprising cellular factors of the invention and apharmaceutically acceptable carrier. Administration can be autologous orheterologous.

If desired, biologically active agents present in conditioned media areincorporated into a polymer scaffold to promote tissue repair, cellsurvival, proliferation in a tissue in need thereof. Polymer scaffoldscan comprise, for example, a porous, non-woven array of fibers. Thepolymer scaffold can be shaped to maximize surface area, to allowadequate diffusion of nutrients and growth factors to a cell of theinvention. Polymer scaffolds can comprise a fibrillar structure. Thefibers can be round, scalloped, flattened, star-shaped, solitary orentwined with other fibers. Branching fibers can be used, increasingsurface area proportionately to volume.

Unless otherwise specified, the term “polymer” includes polymers andmonomers that can be polymerized or adhered to form an integral unit.The polymer can be non-biodegradable or biodegradable, typically viahydrolysis or enzymatic cleavage. The term “biodegradable” refers tomaterials that are bioresorbable and/or degrade and/or break down bymechanical degradation upon interaction with a physiological environmentinto components that can be metabolized or excreted, over a period oftime from minutes to three years, preferably less than one year, whilemaintaining the requisite structural integrity. As used in reference topolymers, the term “degrade” refers to cleavage of the polymer chain,such that the molecular weight stays approximately constant at theoligomer level and particles of polymer remain following degradation.

Materials suitable for polymer scaffold fabrication include polylacticacid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA),polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA),polydioxanone, polygluconate, polylactic acid-polyethylene oxidecopolymers, modified cellulose, collagen, polyhydroxybutyrate,polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid),polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyaminoacids, polyorthoesters, polyacetals, polycyanoacrylates, degradableurethanes, aliphatic polyester polyacrylates, polymethacrylate, acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinylimidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinylalcohol, Teflon®, nylon silicon, and shape memory materials, such aspoly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys, and oligo(ε-caprolactone)diol as switchingsegment/oligo(p-dioxyanone)diol as physical crosslink. Other suitablepolymers can be obtained by reference to The Polymer Handbook, 3rdedition (Wiley, N.Y., 1989).

Methods for Evaluating Therapeutic Efficacy

In one approach, the efficacy of the treatment is evaluated bymeasuring, for example, the biological function of the treated organ(e.g., cardiac cell function). Such methods are standard in the art andare described, for example, in the Textbook of Medical Physiology, Tenthedition, (Guyton et al., W.B. Saunders Co., 2000). In particular, amethod of the present invention, increases the biological function of atissue or organ by at least 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, or even by as much as 300%, 400%, or 500%. Preferably,the tissue is cardiac tissue and, preferably, the organ is heart.

In another approach, the therapeutic efficacy of the methods of theinvention is assayed by measuring an increase in cell number in thetreated or transplanted tissue or organ as compared to a correspondingcontrol tissue or organ (e.g., a tissue or organ that did not receivetreatment). Preferably, cell number in a tissue or organ is increased byat least 5%, 10%, 20%, 40%, 60%, 80%, 100%, 150%, or 200% relative to acorresponding tissue or organ. Methods for assaying cell proliferationare known to the skilled artisan and are described, for example, inBonifacino et al., (Current Protocols in Cell Biology Loose-leaf, JohnWiley and Sons, Inc., San Francisco, Calif.). For example, assays forcell proliferation may involve the measurement of DNA synthesis duringcell replication. In one embodiment, DNA synthesis is detected usinglabeled DNA precursors, such as [3H]-Thymidine or5-bromo-2*-deoxyuridine [BrdU], which are added to cells (or animals)and then the incorporation of these precursors into genomic DNA duringthe S phase of the cell cycle (replication) is detected (Ruefli-Brasseet al., Science 302(5650):1581-4, 2003; Gu et al., Science 302(5644):445-9, 2003).

In another approach, efficacy is measured by detecting an increase inthe number of viable cells present in a tissue or organ relative to thenumber present in an untreated control tissue or organ, or the numberpresent prior to treatment. Assays for measuring cell viability areknown in the art, and are described, for example, by Crouch et al. (J.Immunol. Meth. 160, 81-8); Kangas et al. (Med. Biol. 62, 338-43, 1984);Lundin et al., (Meth. Enzymol. 133, 27-42, 1986); Petty et al.(Comparison of J. Biolum. Chemilum. 10, 29-34, 0.1995); and Cree et al.(AntiCancer Drugs 6: 398-404, 1995). Cell viability can be assayed usinga variety of methods, including MTT(3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop,Bioorg. & Med. Chem. Lett. 1: 611, 1991; Cory et al., Cancer Comm. 3,207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays forcell viability are also available commercially. These assays include butare not limited to CELLTITER-GLO® Luminescent Cell Viability Assay(Promega), which uses luciferase technology to detect ATP and quantifythe health or number of cells in culture, and the CellTiter-Glo®Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH)cytotoxicity assay (Promega).

Alternatively, or in addition, therapeutic efficacy is assessed bymeasuring a reduction in apoptosis. Apoptotic cells are characterized bycharacteristic morphological changes, including chromatin condensation,cell shrinkage and membrane blebbing, which can be clearly observedusing light microscopy. The biochemical features of apoptosis includeDNA fragmentation, protein cleavage at specific locations, increasedmitochondrial membrane permeability, and the appearance ofphosphatidylserine on the cell membrane surface. Assays for apoptosisare known in the art. Exemplary assays include TUNEL (Terminaldeoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assays,caspase activity (specifically caspase-3) assays, and assays forfas-ligand and annexin V. Commercially available products for detectingapoptosis include, for example, Apo-ONE® Homogeneous Caspase-3/7 Assay,FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San Diego, Calif.), theApoBrdU DNA Fragmentation Assay (BIOVISION, Mountain View, Calif.), andthe Quick Apoptotic DNA Ladder Detection Kit (BIOVISION, Mountain View,Calif.).

Methods for Evaluating Cardiac Function

Compositions of the invention may be used to enhance cardiac function ina subject having reduced cardiac function. Methods for measuring thebiological function of the heart (e.g., contractile function) arestandard in the art and are described, for example, in the Textbook ofMedical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co.,2000). In the invention, cardiac function is increased by at least 5%,10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relativeto the cardiac function present in a naturally-occurring, correspondingtissue or organ. Most advantageously, cardiac function is enhanced ordamage is reversed, such that the function is substantially normal(e.g., 85%, 90%, 95%, or 100% of the cardiac function of a healthycontrol subject). Reduced cardiac function may result from conditionssuch as cardiac hypertrophy, reduced systolic function, reduceddiastolic function, maladaptive hypertrophy, heart failure withpreserved systolic function, diastolic heart failure, hypertensive heartdisease, aortic and mitral valve disease, pulmonary valve disease,hypertrophic cardiomyopathy (e.g., hypertrophic cardiomyopathyoriginating from a genetic or a secondary cause), post ischemic andpost-infarction cardiac remodeling and cardiac failure.

Any number of standard methods are available for assaying cardiovascularfunction. Preferably, cardiovascular function in a subject (e.g., ahuman) is assessed using non-invasive means, such as measuring netcardiac ejection (ejection fraction, fractional shortening, andventricular end-systolic volume) by an imaging method suchechocardiography, nuclear or radiocontrast ventriculography, or magneticresonance imaging, and systolic tissue velocity as measured by tissueDoppler imaging. Systolic contractility can also be measurednon-invasively using blood pressure measurements combined withassessment of heart outflow (to assess power), or with volumes (toassess peak muscle stiffening). Measures of cardiovascular diastolicfunction include ventricular compliance, which is typically measured bythe simultaneous measurement of pressure and volume, early diastolicleft ventricular filling rate and relaxation rate (can be assessed fromechoDoppler measurements). Other measures of cardiac function includemyocardial contractility, resting stroke volume, resting heart rate,resting cardiac index (cardiac output per unit of time [L/minute],measured while seated and divided by body surface area [m²])) totalaerobic capacity, cardiovascular performance during exercise, peakexercise capacity, peak oxygen (O₂) consumption, or by any other methodknown in the art or described herein. Measures of vascular functioninclude determination of total ventricular afterload, which depends on anumber of factors, including peripheral vascular resistance, aorticimpedance, arterial compliance, wave reflections, and aortic pulse wavevelocity, Methods for assaying cardiovascular function include any oneor more of the following: Doppler echocardiography, 2-dimensionalecho-Doppler imaging, pulse-wave Doppler, continuous wave Doppler,oscillometric arm cuff, tissue Doppler imaging, cardiac catheterization,magnetic resonance imaging, positron emission tomography, chest X-ray, Xray contrast ventriculography, nuclear imaging ventriculography,computed tomography imaging, rapid spiral computerized tomographicimaging, 3-D echocardiography, invasive cardiac pressures, invasivecardiac flows, invasive cardiac cardiac pressure-volume loops(conductance catheter), non-invasive cardiac pressure-volume loops.

Kits

Compositions comprising a cell of the invention (e.g., a cardiac ormesenchymal stem/progenitor cell that expresses or is primed with CTGF,insulin, or IGF-1) or a composition comprising biologically activeagents (e.g., CTGF, insulin, or IGF-1) is supplied along with additionalreagents in a kit. The kits can include instructions for the treatmentregime, reagents, equipment (test tubes, reaction vessels, needles,syringes, etc.) and standards for calibrating or conducting thetreatment. The instructions provided in a kit according to the inventionmay be directed to suitable operational parameters in the form of alabel or a separate insert. Optionally, the kit may further comprise astandard or control information so that the test sample can be comparedwith the control information standard to determine if whether aconsistent result is achieved.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1. Conditioned Medium from Human Stromal Cells InducedRat CPC Proliferation

Conditioned medium (CdM) was collected from human mesenchymal stem cells(MSCs), p75MSCs, and dermal fibroblasts. CdM from each of the cell typessupported CPC proliferation (FIG. 1A). In contrast, the number of CPCsincubated in CdM vehicle (serum-free α-MEM (SFM)) gradually decreased(FIGS. 1A and 1B). A concentration-dependent increase in CPC number wasobserved when CPCs were incubated in 10×-concentrated CdM (10×CdM) fromMSCs or p75MSCs (FIG. 2A). To confirm DNA synthesis and active cellcycle status, incorporation of BrdU into CPCs was quantified 24 hrsafter exposure to CdM. The percentage of BrdU-positive CPCs in CdM fromMSCs, p75MSCs or fibroblasts was significantly greater than that forSFM-treated CPCs (FIG. 1B). Immunoblotting demonstrated that Ki67 wasexpressed in CPCs treated with CdM but not in CPCs treated with serumfree medium (FIG. 1B). In contrast to its effects on CPCs, CdM from MSCsor p75MSCs did not support the proliferation of adult rat cardiacfibroblasts (FIG. 1C).

Example 2. CdM from Human MSCs Activated STAT3 and Akt in CPCs

STAT3 activation is important for the self-renewal of ES cells, adultHSCs, and adult neural stem cells. Accordingly, levels ofphosphorylated-STAT3 (p-STAT3) in CPCs exposed to CdMs from MSCs,p75MSCs, or fibroblasts were assayed. Levels of p-STAT3 weresignificantly higher in CPCs at 1 and 2 days after CdM treatmentcompared with SFM treatment (FIG. 3A). Immunocytochemistry demonstratedthat p-STAT3 localized to the nuclei of CPCs treated with CdM (FIG. 3B).In addition to p-STAT3, phosphorylation of Akt (p-Akt) was observedafter incubation of CPCs in CdM from each of the stromal cell types(FIG. 3C). However, p-Akt in CPCs incubated in SFM alone was alsoobserved (FIG. 3C). Notably, auto-phosphorylation of signaling moleculesaffecting cell survival, such as Akt, ERK1/2, and mTOR occurs in diversecell types during serum or nutrient deprivation.

To determine whether p-STAT3 and/or p-Akt mediated the effects of CdM onCPCs, CPCs were treated with pharmacological inhibitors. AG490, aJak2/STAT3 pathway inhibitor, reduced the number of CPCs treated withMSC CdM in a dose-dependent manner: control (CdM+DMSO), 100±1.5%; 1 μM,96.3±0.9%; 5 μM, 89.5±0.9%; 10 μM, 43.4±2.4% (cell number ratio tocontrol cell number [121,863 cells], mean±SEM, n=3 to 6). The inhibitoryeffect of AG490 (10 μM) was also observed for CPCs treated with CdM fromp75MSCs or fibroblasts (FIG. 3D). LY294002, a phosphatidylinositol3-kinase (PI3K)/Akt pathway inhibitor (10 μM), decreased CPC number inCdM, but to a lesser extent than AG490 (FIG. 2E). Combined treatmentwith AG490 and LY294002 (10 μM, each) was most effective in reducing CPCnumber in CdM (FIG. 3E). Incubation of CPCs in “Stattic” (10 μM), ahighly specific STAT3 inhibitor (Schust et al., Chem Biol 2006;13(11):1235-1242), confirmed the role of STAT3 activation inCdM-mediated effects on CPC proliferation (FIG. 2B). PD98059, an ERKinhibitor, did not diminish CPC proliferation in CdM.

Insulin and Insulin-like growth factor 1 (IGF-1) bind to tyrosine kinaseholoreceptors and promote cell survival and proliferation by signalingthrough the PI3K/Akt and Ras/MAP kinase pathways. Partial functionalredundancy for Insulin and IGF-1 signaling is evidenced by signalingthrough receptor heterodimers IR/IGF-1R and bidirectional cross-talkbetween ligands/receptors. CSCs/CPCs did not proliferate in the absenceof medium supplements containing Insulin (e.g.Insulin-Transferrin-Selenium [ITS] or fetal calf serum), even when inthe presence of other mitogenic components from CSC/CPC growth mediumsuch as LIF, EGF or bFGF (FIG. 2C). This suggested that CdM from humanstromal cells may contain Insulin, IGF-1, or both.

Example 3. CdM Protected CPCs Exposed to Hypoxia

Because cells transplanted to the heart after myocardial infarction (MI)may encounter hypoxic environments, it was examined whether CdM couldprotect CPCs during exposure to 1% oxygen for 48 hrs ex vivo. CdM fromMSCs and p75MSCs significantly promoted the survival of CPCs comparedwith SFM (FIG. 4A). Compared with cell survival in CPC growth medium(positive control; 100±6.2%), survival of CPCs in SFM was 59.9±3.2%while that of CPCs incubated in MSC CdM was 90.3±6.0% (P<0.05 vs SFM)and p75MSCs CdM was 93±1.5% (P<0.0001 vs SFM). Survival of CPCsincubated in 1×CdM from 1 of 2 MSC donors tested and both p75MSC donorstested was not significantly different from that in growth medium (MSCdonor 4, P=0.66; p75MSC donor 1, P=0.33; p75MSC donor 3, P=0.37).Addition of STAT3 inhibitor abolished CdM-mediated protection of CPCsunder hypoxic conditions (AG490, FIG. 4B; Stattic, FIG. 4C).

Example 4. CPCs Incubated in CdM Retained their MultipotentDifferentiation Capacity

Control CPCs cultured in CPC growth medium were negative for α-smoothmuscle actin and von Willebrand Factor staining, whereas about 60%expressed α-sarcomeric actin (FIGS. 5A and 5B, left). In contrast,clones of CPCs exposed to 1×CdM for 4 days stained positively forα-sarcomeric actin, α-smooth muscle actin, and von Willebrand Factor(FIGS. 5A and 5B, right). After 4 days in CdM, CPC-derived cells nolonger expressed the CSC antigen, c-Kit, indicating progress towarddifferentiation.

Example 5. Intra-Arterial Infusion of p75MSC CdM Improved CardiacFunction after MI

Based on the results with CdM and cultured CPCs, it was examined whetherinfusion of CdM after MI would increase the number of endogenous CPCs toimprove cardiac repair and function. MI was produced in C57bl6/J mice bypermanent ligation of the left anterior descending coronary artery(LAD). The following day, mice were randomized to receive treatment withintra-arterial infusion (left ventricle lumen) of 30×p75MSC CdM orvehicle (SFM). Mice from each group were euthanized at 1 day or 1 weekafter treatment. Hearts obtained 1 day after treatment were sectionedand processed for TUNEL and immunohistochemistry to detect c-Kit. TUNELassays showed that CdM infusion significantly reduced cardiacapoptosis/necrosis (SFM, 17.2±8.1%; CdM, 3.7±2.2%; P<0.05, FIGS. 6A-6C).Intra-muscular injection of porcine MSCs from bone marrow, but not theirconditioned medium, was reported to increase the number of c-Kit⁺ cellsin the hearts of pigs after MI. After intra-arterial infusion of CdMfrom human p75MSCs, rare c-Kit⁺ cells were detected in heart sectionsfrom both CdM- and SFM-treated mice. However, because c-Kit⁺ cell numberwas variable and did not appear to differ between mice that received CdMor SFM, c-Kit⁺ cells were not quantified. At 1 week after treatment,echocardiographic measurements demonstrated significantly improvedcardiac function in mice that received CdM compared with SFM (FIGS. 7and 8 ). Biochemical assays for residual myocardial Creatine Kinase (CK)activity in left ventricular (LV) homogenates indicated that CdM- andSFM-treated mice had similar size infarcts at 1 week after MI (AnteriorLV, SFM, 4.66±0.40; CdM, 5.00±0.43, P=0.21; Posterior LV, SFM,6.59±0.32; CdM, 6.34±0.33, data expressed as IU CK/mg protein, mean±SD,P=0.23; SFM, n=6; CdM, n=5).

Example 6. Priming of CSCs with CdM from p75MSCs Improved Graft Successafter MI

Based on the observations with p75MSC CdM and its ability to increaseproliferation and survival of cultured CSCs/CPCs, it was determinedwhether prior exposure of CSCs to p75MSC CdM could foster the graftingof CSCs to the injured heart. MI was produced in Fischer rats bypermanent ligation of the LAD. One day after MI, syngeneic GFP-positiverat CSCs were primed for 30 min. on ice in p75MSC CdM (30×CdM, n=6 rats)or vehicle (SFM, n=5 rats). The chest wall was re-opened and rats wererandomized to treatment with co-injections of CSCs/CdM or CSCs/SFM(125,000 cells/5 μl injection, 2 sub-epicardial injections, 1 per borderzone). At 1 week after MI, rats were euthanized and their hearts wereprocessed as frozen serial-sections from apex to base. For each heart,GFP⁺ cells were quantified in the tissue section with the most GFP⁺cells. For 2 rats in CSCs/SFM co-injection group, GFP⁺ cells were notdetected. Furthermore, hearts from CSC/SFM rats with the highest levelof engraftment contained less than 40 GFP⁺ cells/section (FIG. 9A). Incontrast, many sections from rats co-injected with CSCs/CdM containedseveral thousand GFP⁺ cells (FIG. 9B-9E). After priming of CSCs inp75MSC CdM, GFP⁺ cells grafted into sub-epicardial locations,proliferated (see Ki67 stain, FIG. 4B), and migrated into zones withinfarction (FIGS. 9C-9G and 10 ). Furthermore, after 1 week, derivativesfrom CSCs primed in CdM engrafted into blood vessel walls asCD31-positive endothelial cells (FIG. 9F). They also generated smoothmuscle cells and myofibroblasts (smooth muscle alpha actin-positive,FIG. 9G). Although they grafted into sub-epicardial locations after MI,GFP⁺ cells derived from vehicle-primed CSCs did not stain for Ki67, andhad not entered myocardial tissue from sub-epicardial tissue at 1 weekafter MI and treatment (FIG. 9A).

Example 7. CdM Contains Human CTGF and Insulin that Activated CSCs/CPCs

To identify factors in p75MSC CdM that promoted the proliferation andsurvival of CPCs, Affymetrix gene expression profiles were examined fromhuman p75MSCs that were freshly sorted from marrow aspirates and fromp75MSCs cultured adherently for 2 passages (Bakondi et al., Mol Ther2009; 17(11):1938-1947). Antibody blocking/neutralization studies forselected secreted factors were carried out with 10×p75MSC CdM and CPCsexposed to hypoxic conditions (1% oxygen, 48 hrs). Under hypoxia,neutralizing antisera specific to human CTGF prevented CdM fromprotecting CPCs (P<0.001, FIG. 11A). Of interest, addition of human CTGFalone to SFM containing 1% BSA significantly induced p-STAT3 in CPCs(see inset, FIG. 11A). By ELISA, 10×p75MSC CdM contained 1.00±0.02 ng/mlhuman CTGF. Although human IGF-1 in p75MSC CdM was not detected, it wasfound that 10×p75MSC CdM contained 8.75±2.7 ng/ml of bovine Insulin.Furthermore, neutralizing antibodies specific to Insulin significantlyreduced CdM-mediated protection of CPCs during hypoxia exposure, albeitnot as much as did blocking CTGF (MTS assay[Abs⁴⁹⁰]: non-specific IgG,0.378±0.021; anti-Insulin, 0.270±0.034; mean±SD, n=4, P<0.05). Additionof recombinant human Insulin to CPCs induced p-Akt in a dose-responsivemanner and increased also CPC survival and proliferation under hypoxicconditions (FIG. 11B). Notably, CPC protection assays with human Insulinor IGF-1 alone (30 ng/ml, each) demonstrated that they were equivalentin their ability to rescue CPCs exposed to hypoxia (control [SFM with 1%BSA]: 9004±12 cells; Insulin: 13,507±1,473 cells; IGF-1, 14,894±559cells; mean±SD, n=3, P=0.24).

CTGF (CCN2, IGFBP8) consists of 4 domains and the C-terminal (4^(th))domain alone was reported to increase cell adhesion and proliferation(Steffen et al., Growth Factors. 1998; 15(3):199-213; Gao et al. J BiolChem. 2004; 279(10): 8848-8855). In experiments with recombinantpeptides, combined treatment with human C-terminal CTGF peptide(CTGF-D4) and Insulin (i.e., alpha chain-beta chain dimer) hadsynergistic effects on CPC survival and proliferation under hypoxicconditions. For example, CTGF-D4 or Insulin alone (1 ng/ml in SFM, each)did not protect CPCs against 48 hr of hypoxia (FIG. 1C). In contrast,addition of both CTGF-D4 and Insulin to SFM (1 ng/ml, each) providedsignificant protection of CPCs against hypoxia (P<0.05, FIG. 1C).CTGF-D4/Insulin-mediated protection of CPCs was enhanced by including 1%BSA as a carrier (P<0.01, FIG. 1C).

Example 8. A Defined Combination of CTGF-D4/Insulin Promoted CSC Graftsafter MI

Having observed synergistic protective effects after CTGF-D4/Insulintreatment of cultured CPCs exposed to simulated ischemia (FIG. 11C), itwas hypothesized that a priming mixture based on the CTGF-D4/Insulinratio found in 30×p75MSC CdM would promote CSC engraftment when using toprime CSCs. One day after MI, GFP CSCs were incubated on ice with SFMcontaining 1% BSA, CTGF-D4 (3 ng/ml), and Insulin (30 ng/ml), or withvehicle (SFM with 1% BSA) for 30 min. prior to co-injection into borderzone areas of rats randomized to treatment (125,000 cells/5 μlinjection, 2 sub-epicardial injections, 1 per border zone). As before,all rats were euthanized 1 week after MI and their hearts were processedas serial sections. Whereas few rats that received co-injections ofCSCs/vehicle had detectable GFP⁺ cells after 1 week (n=1/7 rats, FIGS.12A and 12B), all rats that received CSCs/CTGF-D4/Insulin exhibited alevel of engraftment consistent with results obtained by priming with30×p75MSC CdM (5/5 rats, FIG. 12B-12C′). Similar to CSCs primed in CdM,CSCs primed in CTGF-D4/Insulin grafted into sub-epicardial locations,proliferated, and provided GFP⁺ CSC derivatives that migrated into hostmyocardium, reaching areas of infarction with few remaining viablemyocytes (FIG. 12B′-12C′).

Example 9. p75-CdM and CTGF-D4 Promoted Signaling Through the WntPathway in Epicardial-Derived Cells (EPDC)

While examining different signaling pathways that might be stimulated byp75-CdM and/or CTGF-D4, it was found that epicardial-derived cells(EPDC) expressed the Wnt co-receptor Lrp5/6 and that both p75-CdM andCTGF-D4 promoted signaling through the canonical Wnt pathway, asevidenced by phosphorylation of GSK3β and accumulation of nuclearβ-catenin (FIGS. 13A and 13B). Wnt signaling occurs when a Wnt ligandassociates with Frizzled (FRZ) and Lrp5/6, a Wnt ligand co-receptor.This results in activation of the cytoplasmic phosphoprotein, Disheveled(DSH) and release of β-catenin from a “destruction complex” made up ofthe scaffold protein, Axin, the tumor suppressor protein adenomatouspolyposis coli (APC), and glycogen synthase kinase 3β (GSK3β). Underthese conditions, GSK3β does not phosphorylate β-catenin; this leads toaccumulation of β-catenin in the cell nucleus where it binds to TCF/LEFfamily transcription factors and activates gene transcription. In theabsence of Wnt ligand or receptor stimulation, β-catenin isphosphorylated by GSK3β, tagged by an E3 ubiquitin ligase, and destroyedby proteasomal degradation.

Furthermore, incubation of EPDCs in Dkk-1, a secreted inhibitor of Lrp6,significantly reduced the protection normally conferred by CTGF-D4 whenEPDCs were incubated under hypoxic conditions (1% oxygen, 48 hr)(p<0.05, FIG. 13C). To determine whether CTGF-D4 interacted with Lrp6 topromote EPDC survival, cell protection assays were performed underhypoxia and with neutralizing antisera to Lrp5/6 or specifically, Lrp6.Both antibodies significantly reduced the protection conferred byCTGF-D4 (p<0.05 for both, FIGS. 13D and 13E).

It was also investigated whether signaling through CTGF-D4/Lrp6 played arole in graft success after MI. A two-color competitive grafting assayfor EPDCs was developed to be able to evaluate inhibition effects (e.g.primed with CTGF-D4 and anti-Lrp6) on a separate aliquot of cells fromthe same isolate that was used for control (e.g. primed withCTGF-D4/Insulin and non-specific IgG). Importantly, labeling of EPDCswith cell-tracking dye did not affect their ability to graft and migrateinto the heart after MI (see distance traveled from subepicardialinjection site at 1 week in FIG. 1F). Adding blocking antisera to Lrp6to the CTGF-D4/Insulin priming mix substantially reduced the number ofgrafted EPDCs when hearts were examined at 1 week after MI (celltransplant 1 day after MI)(FIGS. 13G and 13H). The difference forcontrol cells (non-specific IgG, red) and anti-Lrp6 cells (green) wasespecially evident at locations distal to the subepicardial graft site(FIG. 13H). Pull-down assays were performed to remove CTGF from p75-CdM(FIG. 13I). In addition, lentiviral transductions of human p75MSCs withshRNAs against CTGF were performed. Several differentpuromycin-selectable CTGF shRNA vectors were tried. One that was notdetrimental to p75MSC growth was identified, and elicited a ˜60%decrease in CTGF levels by ELISA (p<0.0001 compared with CdM fromcontrol cells transduced with a scrambled shRNA vector, FIG. 13J).

These results show that the Wnt pathway is involved in the effects ofp75-CdM and CTGF-D4 on epicardial-derived cells.

Example 10. CTGF-D4 Promoted Adhesion to Fibronectin and EMT-LikeDifferentiation in Epicardial-Derived Cells (EPDC)

In ex vivo adhesion assays with EPDCs, CTGF-D4 (40 ng/ml) promotedadhesion of EPDCs to fibronectin (FIG. 14A). Exposure to CTGF-D4 alsopromoted EMT-like differentiation in EPDCs, increasing protein levels ofsmooth muscle actin (SMA), the adhesion/migration mediator Thy-1 (CD90),and the Endothelin Type B receptor (ETrB)(FIG. 14B). Levels of VonWillebrand Factor, an endothelial-specific cell marker, did not seem tobe affected by CTGF-D4 (FIG. 14B). Notably, co-incubation of EPDCs inCTGF-D4 and anti-Lrp6 strongly reduced SMA, CD90, and ETrB proteinlevels (FIG. 14C). Furthermore, co-incubation in CTGF-D4 and anti-Lrp6markedly increased Keratin protein expression (FIG. 14C). Without beingbound to a particular theory, this may indicate a reversal of EMT backtoward an epithelial (mesothelial) state. Multiple neutralizing antiseraand pharmacological inhibitors were screened for a variety of cellsurface receptors to interrogate their potential role in CTGF-D4signaling in EPDCs. Drug antagonists to ETrB and FGFR1 significantlyreduced cell protection conferred by CTGF-D4 under hypoxic conditions(ETrB antagonist, p<0.01; FGFR1 antagonist, p<0.05; FIGS. 14D and 14E).

Example 11. Epicardial-Derived Cell (EPDC) CdM Provided VascularProtection In Vivo and In Vitro

To examine EPDC CdM-mediated vascular protection, cardiac endothelialcell protection assays were performed under conditions of simulatedischemia: low glucose medium and 1% oxygen for 24 or 48 hrs. Comparedwith incubation in MEM (vehicle control), unconcentrated CdM (1×CdM)generated from EPDC protected cardiac endothelial cells fromhypoxic/ischemic injury for 24 hr in this assay. CdM concentrated10-fold (10×CdM) provided a greater level of cell protection than did1×CdM, protecting coronary artery endothelial cells, microvascularendothelial cells and aortic endothelial cells for 48 hr.

To determine whether EPDC CdM could provide vascular protection in vivo,adult male Fischer rats underwent 2 hr of ischemia followed by 24 hr ofreperfusion. They were then treated with either MEM (vehicle control,MEM) or 30×EPDC CdM at the time of reperfusion. Two hours prior toharvesting the heart, each rat was injected with 0.5 ml of FITC-albumin(5 mg/ml) through the tail vein. Hearts were then harvested andhomogenized. Twenty-four hours after ischemia/reperfusion we quantifiedthe amount of FITC extravasation in each treated animal by normalizingit to extravasation in sham-operated rats that were also injected withFITC-albumin. Heart homogenates from EPDC CdM-treated rats contained37.8% greater FITC fluorescence that those from rats that receivedvehicle. The amount of extravasated FITC-albumin in the vehicle-treatedgroup was 527.4%±109.33 of sham (n=5), while in the CdM-treated group itwas 359.7724%±78.82 of sham (n=5)(FIGS. 15A and 15B). Thus, EPDC CdMcontains factors that promote blood vessel integrity early afterreperfusion.

To understand the effects of EPDC CdM on blood vessels, immunoblottingwas performed on the soluble fraction from left ventricle homogenates.VE-Cadherin plays a key role in maintaining endothelial barrierintegrity, and the level of phosphorylated VE-Cadherin (pVE-Cadherin) isa marker for increased vascular permeability. At 1 day after MI,reperfusion, and treatment, the level of pVE-Cadherin was significantlyhigher in the MEM group of animals than in the EPDC CdM group (p≤0.05,n=3; FIGS. 15C and 15D). To examine the relative effects of EPDC CdMtreatment on vascular endothelial cells and smooth muscle cells, thelevels of CD31 (PECAM1, endothelial marker) and smooth muscle alphaactin (SMA) were compared in LV homogenates from vehicle- and EPDCCdM-treated groups. Consistent with endothelial cell survival,significantly higher levels of CD31 were observed in animals treatedwith EPDC CdM in comparison with vehicle controls (p<0.05, n=3; FIGS.15C and 15D). In contrast to the CD31 results, equal amounts of solubleSMA in vehicle- and EPDC CdM-treated animals were observed (FIG. 15C).These data indicate that EPDCs secrete a factor or factors that reducevascular permeability and increase endothelial cell survival.

Materials and Methods Preparation and Isolation of Human StromalProgenitor Cells and Fibroblasts.

Human MSCs, p75MSCs and dermal fibroblasts were prepared with protocolsapproved by an Institutional Review Board (Bakondi et al., Mol Ther2009; 17(11):1938-1947). To obtain MSCs, bone marrow aspirates weretaken from the iliac crest of healthy adult donors. Mononuclear cellswere isolated with the use of density gradient centrifugation(Ficoll-Paque, Amersham Pharmacia Biotech) and resuspended in completeculture medium consisting of Alpha-MEM (GIBCO/BRL, Grand Island, N.Y.);17% FBS (Atlanta Biologicals, Norcross, Ga.); 100 units/ml penicillin(GIBCO/BRL); 100 μg/ml streptomycin (GIBCO/BRL); and 2 mM L-glutamine(GIBCO/BRL). Cells were plated in 20 ml of medium in a 150 cm² culturedish and incubated in a humidified incubator (Thermo Electron, FormaSeries II, Waltham, Mass.) with 95% air and 5% CO₂ at 37° C. After 24hr, nonadherent cells were removed. Adherent cells were washed twicewith PBS and incubated with fresh medium. The primary adherent cellswere cultured and propagated.

To obtain p75MSCs, bone marrow stem/progenitor cells were isolated byMACS using antibodies against the p75LNGFR. Freshly isolated bone marrowmononuclear cells from the Ficoll gradient were resuspended in 0.4 ml ofPBS containing 0.5% bovine serum albumin and 2 mM EDTA. After addingmouse anti-human p75LNGFR antibody conjugated to magnetic beads (CD271,Miltenyi Biotech, Auburn, Calif.), the sample was incubated for 30 min.at 4° C., and then applied to a magnetic column (LS Column; MiltenyiBiotech). The bound fraction was eluted with 5 ml of MACS buffer and thecells were concentrated by centrifugation at 1000×g for 8 min. Afterre-suspension, the entire isolate was cultured in complete culturemedium. MSC-like cells appeared as small colonies after about 1 week,and the cells were expanded.

Isolation and Culture of Adult Rat Cardiac Stem/Progenitor Cells.

Adult CSCs were isolated from the ventricles of Fischer 344 rats andlabeled with retroviral vector for GFP (Bakondi et al., Mol Ther 2009;17(11):1938-1947). CSCs were cultured as floating spheres in DMEM/F12supplemented with bFGF (10 ng/ml), EGF (20 ng/ml), LIF (10 ng/ml) andITS. To grow adherent CPCs, CSCs were plated at 500 cells/cm² andcultured in CSC medium supplemented with 2% FBS (CPC growth medium).

Isolation and Culture of Adult Rat Cardiac Fibroblasts.

Ventricular fibroblasts were prepared with protocols approved by anInstitutional Animal Care and Use Committee. Ventricular fibroblastswere isolated from hearts of adult Sprague-Dawley rats. The hearts wereminced and enzymatically-dissociated into single cell suspension.Non-myocytes were separated by discontinuous density gradientcentrifugation and cultured in DMEM/F-12 supplemented with 10% FCS.Passage 2 cells were used for experiments.

Preparation of Serum Free Conditioned Medium (CdM).

Passage 4 to 8 human MSCs, p75MSCs or dermal fibroblasts were culturedto 80 to 90% confluence in 150 cm² dishes with complete culture medium(Bakondi et al., Mol Ther 2009; 17(11):1938-1947). To generate CdM,cells were washed twice with PBS and incubated with 20 mls of freshserum-free α-MEM in standard conditions without any supplements orgrowth factors for 48 hrs. Medium was then collected, filtered, andstored at −80° C. For some experiments, CdM was concentrated up to 10-or 30-fold with the use of a Labscale™ TFF diafiltration system(Pellicon XL 5 kDa cut-off filters, Millipore, Bedford, Mass.).

Short-Term Priming of CSCs.

CSCs were cultured as spheres in serum-free CSC growth medium. CSCspheres were trypsinized and centrifuged at 1000×g for 8 min. Afterre-suspension in 1×PBS, cells were passed through a 40 micron filter toisolate single CSCs (cell strainer, Fisher Scientific). Cells werecounted on a hemocytometer, centrifuged again, and re-suspended in30×p75MSC CdM or Alpha-MEM (CdM vehicle control), or CTGF-D4 (3ng/ml)/Insulin (30 ng/ml)/1% BSA, or 1% BSA in Alpha-MEM (vehiclecontrol for recombinant peptides). CSCs were incubated in the aboveconditions for 30 minutes on ice prior to sub-epicardial injection.

Myocardial Infarction Surgery and CSC Transplantation in Rats.

Fischer 488 rats (males, 7 weeks of age) were weighed, shaved,anesthetized under 4% isoflurane, and endotracheally-intubated. Ratswere ventilated at a respiration rate of 65 beats per min under a peakinspiration pressure of 15 cm H₂O (Kent Scientific). Body temperaturewas maintained at 37° C. with a heating pad (Gaymar). Through a dermalincision, a blunt dissection of the fascia was performed and theintercostal muscles were separated. The heart was exposed by retractionof the pericardium to expose the LAD. The LAD was occluded with a 6-0nylon suture and occlusion was confirmed by blanching of the anteriorfree wall of the LV. The animals were allowed to recover off theventilator.

After 24 hours, rats were re-intubated, ventilated, and the chest wallwas re-opened. Hearts were exposed to reveal the border zones of theinfarct. For each rat, we performed 2 sub-epicardial injections of CSCs(5 ul each, one per border zone) with a 30 gauge Hamilton syringe. Theneedle was introduced tangentially to the wall of the LV and with thebevel facing upward. The syringe was advanced only as far as the beveledge to access the sub-epicardial surface of the heart and so as nottarget the underlying myocardium of the LV. After the injections, thechest wall was closed and rats recovered for 7 days prior toeuthanization.

Cell Culture in CdM and Evaluation of Cell Number.

Adult rat CPCs and cardiac fibroblasts were plated at 500 cells/cm² andcultured in their respective growth mediums. Three days after plating,the medium was removed, the wells were washed twice with PBS, and thecells were then exposed to CdM or to fresh serum-free medium(Alpha-MEM). For time course proliferation studies, CdM and serum-freemedium were changed every 2 days. In signal transduction inhibitorstudies, the following pharmacological inhibitors were used: AG490,inhibitor of Jak2/STAT3 pathway; Stattic, inhibitor of STAT3; LY294002,inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt pathway; andPD98059, inhibitor of extracellular signal-regulated kinase (ERK). Allof the inhibitors were purchased from Calbiochem (Darmstadt, Germany)and were dissolved in dimethyl sulfoxide (DMSO). CPCs were cultured inCdM with the inhibitors or with the equivalent volume of DMSO as acontrol for 48 hrs. In cell protection studies, 3 days after plating,medium was replaced with either the CdM or serum-free medium and thecells were exposed to hypoxia in a specialized incubator (1% oxygen) for48 hrs. The hypoxia incubator was a model that measured both CO₂ and O₂(Thermo Electron, Forma Series II, model 3130). Oxygen was maintained at1% by the injection of nitrogen gas and was monitored continuously.

Cell numbers were quantified by the fluorescent labeling of nucleicacids (CyQuant dye; Molecular Probes, Carlsbad, Calif.) and with amicroplate fluorescence reader (FL×800; Bio-Tek Instruments Inc.,Winooski, Vt.) set to 480 nm excitation and 520 nm emission. Eachexperiment was repeated a minimum of 3 times.

Immunocytochemistry.

CPCs were fixed with 4% paraformaldehyde in 1×PBS. Non-specific bindingwas limited by 1 hour incubation in PBS containing 5% goat serum and0.4% triton X-100. Primary antibodies were incubated overnight at 4° C.After washing 3×5 min with PBS, secondary antibody that was diluted1:1000 (Alexa 594, Molecular Probes) was applied for 1 hr at roomtemperature (RT). After 3×5 min washes, slides were mounted withVectashield containing DAPI (Vector Laboratories, Burlingame, Calif.).Epifluorescence images were taken using a Leica DM6000B microscopeequipped with a CCD camera (Leica DFC350Fx) and FW4000 software. Theprimary antibodies for immunocytochemistry were as follows:phospho-STAT3 (Tyr705, 1:50, Cell signaling, Danvers, Mass.);α-sarcomeric actin (1:500, Sigma); α-smooth muscle actin (1:800, Sigma);and von Willebrand factor (1:100, Chemicon, Temecula, Calif.). Forquantification of differentiation, cells positive for α-sarcomericactin, α-smooth muscle actin and von Willebrand factor and total cellswere counted at least in three fields per slide. The percentage ofpositive cells was calculated for each slide (n=3 in each group).

DNA Replication Assay.

Three days after the plating, CSCs were cultured in growth medium, CdMor serum-free medium for 24 hr, and BrdU (BD Biosciences) was added at afinal concentration of 10 μM. Immunocytochemistry was carried out withBrdU antibody (Sigma) and BrdU-positive cell numbers were quantified asdescribed herein above.

Immunoblotting.

Cells were lysed in a buffer that consisted of 0.1% sodium dodecylsulphate (SDS) and complete protease inhibitor cocktail (Roche, Basel,Switzerland) in PBS. Protein concentration was determined by the DCprotein assay (Biorad, Hercules, Calif.). Twenty μg of protein wasseparated by SDS-PAGE. After electrophoresis, the gels wereelectroblotted to polyvinylidene difluoride (PVDF) membranes. Allelectrophoresis and electroblotting used Novex reagents and systems(Invitrogen, Carlsbad, Calif.). The blots were blocked for 1 hr at RT in5% nonfat dry milk in PBS with 0.1% Tween 20 (PBST), washed 3×5 min inPBST, and incubated in primary antibodies in PBST with 5% BSA overnightat 4° C. After 3×5 min washes in PBST, the blots were incubated insecondary antibody conjugated to horseradish peroxidase conjugate(1:2000, Sigma) in PBST for 1 hr at RT. Unbound secondary antibody wasremoved and positive bands were detected with a chemiluminescentreaction. The primary antibodies for immunoblotting were Ki67 (cloneSP6, 1:200, Abcam, Cambridge, Mass.); p-STAT3 (Tyr705, 1:1000), totalSTAT3 (1:1000), p-Akt (Ser 473, 1:1000)(Cell signaling); and β-actin(1:5000, Sigma).

ELISAs for IGF-1, Insulin, and CTGF.

For assay of human IGF-1, lx p75 CdM was assayed by ELIAS usingcommercial ELISA reagents according to manufacturer's protocol (R and DSystems). For assay of Insulin and CTGF, high protein-binding plateswere incubated with 1 or 10×p75 CdM overnight at room temperature tocapture antigens from CdM. Wells were then washed with mild detergent(0.05% Tween-20 in PBS) followed by blocking with 1% BSA in PBS for 1hour. After blocking buffer was thoroughly washed off from the wells,samples were incubated with 100 μl of biotin-conjugated polyclonalantibody to CTGF at 5 μg/100 μl (Peprotech) for 2 hours at roomtemperature. Polyclonal mouse anti-Insulin antibody (Santa Cruz) wasincubated for 2 hours at room temperature, followed by 3 washes withwash buffer. The wells were then incubated in anti-mouse biotinconjugated IgG (Sigma Aldrich) for 2 hours at room temperature. Afterwashing in wash buffer 3 times, samples for CTGF and Insulin ELISA wereincubated in Streptavidin conjugated HRP (1:2000) for 2 hrs at roomtemperature, followed by washing and addition of 100 μl of substrateABTS (Thermo Scientific; #37615) for 20 minutes. Absorbance was measured(450 nm) on a Synergy HT plate reader.

Myocardial Infarction Surgery in Mice.

Male mice at 8-10 weeks of age underwent permanent ligation of the LeftAnterior Descending Coronary Artery (LAD) to induce myocardialinfarction (C57bl6 mice, Taconic, Hudson, N.Y.). Mice were not includedin the study if they did not survive the initial MI surgery, did notachieve a successful MI (blanching observed at time of treatment), ordied during treatment application. Following all procedures, mice weregiven analgesia (buprenorphine, 0.05-0.1 mg/kg i.p.) and monitored forsigns of distress until termination of the study. All procedures weredone in accordance with protocols approved by an Institutional AnimalCare and Use Committee.

For permanent LAD ligation surgery, mice were anesthetized with 2-4%Isoflurane, shaved, weighed, intubated, and then maintained for theduration of the procedure on a sterile surgical field with the use of amechanical ventilation system (MiniVent, Harvard Apparatus, Holliston,Mass.). Throughout the surgery and during the recovery period, bodytemperatures were maintained with a heated water pad system (GaymarT-Pump TP-500, Gaymar Industries, Orchard Park, N.Y.). Viewing the chestthrough a dissecting microsope (Stemi 2000-C, Carl Zeiss MicroImaging,Thornwood, N.Y.) a dermal incision was made, the underlying fascia wereremoved, and the thoracic musculature was retracted to expose the leftribcage. Next the intercostal muscles were retracted and the outer(parietal or visceral) pericardium was removed to expose the LAD. TheLAD was then ligated (2.0-3.0 mm from left atrial apex) with 8.0 nylonsuture (Henry Schein, Melville, N.Y.) and blanching within themyocardium of the left ventricle was noted. The intercostals wererejoined with a 6.0 nylon suture (Henry Schein), the lungs werereinflated, and overlying dermis rejoined with a 6.0 nylon suture. Allmice were recovered to an ambulatory state prior to any subsequenttreatment procedure. Survival after the MI surgery was >90%.Sham-operated mice underwent all procedures except that the suture wasplaced under the LAD but was not ligated.

Infusion of p75MSC CdM after MI in Mice.

To evaluate p75 CdM treatment in an unbiased manner, all animals wererandomized to treatment (after LAD ligation). Following 24 hour recoveryof an animal after the first surgery, the mouse was then againanesthetized, intubated, and the chest opened. Once the intact sutureand area of blanching were confirmed, 30×p75 CdM (200 μL) or vehicle(Alpha-MEM, 200 μL) warmed to 37° C. was delivered to the entirecardiovascular arterial tree by injecting the solution into the lumen ofthe left ventricle (LV). Injections were performed slowly (over a periodof 1 minute) with a 30.5 gauge needle inserted below the great cardiacvein (LV apex) at an angle 45° to the myocardium. Following treatmentwith either CdM or vehicle, the needle was removed and the intercostalswere rejoined using 6.0 chromic gut suture (Ethicon, Johnson andJohnson, Inc., Livingston, UK), lungs then reinflated, and overlyingdermis rejoined with 6.0 nylon suture. All mice were then recovered toan ambulatory state and transferred to the vivarium for the remainingduration of the experiment.

Echocardiography.

Two dimensional, Doppler, and M-Mode echocardiography was performed witha Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, ON,Canada). Data were recorded from sham-operated mice, vehicle-treatedmice with MI, and CdM-treated mice with MI while under isofluraneanesthesia. All left ventricular dimensions in systole and diastole weremeasured from M-mode images obtained at the mid-papillary muscle level.Echocardiographic data were coded for unbiased measurements anddetermination of ECHO wall motion scores. ECHO scores were determinedfrom functional assessment of 13 segments with a model based on theAmerican Society of Echocardiography 17 segment model. Systolic wallmotion scores were assigned to 4 quadrants each of 3 short axis segmentstaken at apical, mid papillary, and basal levels, with an additionalsegment at the apex (13 total). Scores were assigned as: 1=normal, >25%motion; 2=hypokinetic, 10-25% motion; 3=akinetic, <10% motion. Pulmonaryarterial Doppler flow velocities and volumes were quantified (Baumann etal. Echocardiography 2008; 25:739-748).

Following ECHO, the mice were killed humanely by exsanguination and thehearts were removed and rinsed in PBS. For CK assays, left ventriculartissue was dissected away from the atria and the aorta, furtherseparated into anterior LV and posterior LV/septum, and immediately snapfrozen by submersion of cryovials in liquid N2. The LV tissues weremaintained at −80° C. until the day of the CK assay (see below).

TUNEL Assay.

TUNEL was performed as reported previously (French et al. FASEB J 2009;23:1177-1185). Quantification of TUNEL-positive cells within zones ofinfarction was performed in an unbiased fashion by a viewer that wasblinded to slide (sample) identity. Cells were counted with Image ProPlus Software as reported previously (French et al. FASEB J 2009;23:1177-1185).

Creatine Kinase Assay.

The remaining creatine kinase (CK) activity in left ventricular tissueswas assessed to determine the extent of infarction in mouse hearts asreported previously (Kjekshus et al. Circ Res 1970; 27:403-414; Zaman etal. Exp Biol Med. 2011; 236:598-603). The loss of CK activity directlyreflects the loss of viable myocardium after MI. The percentage of leftventricle with infarction was calculated based on observed total LV CKactivity (IU/mg protein) in left ventricles of normal hearts withoutinfarction. The percent of MI=100×[NL CK-LV CK]/Δ, where NL CK is theamount of CK in tissue from normal LV (IU/mg of soluble protein), LV CKis total remaining CK activity in the LV after MI (IU/mg solubleprotein), and A is the difference between the amount of CK in normalzones of myocardium and in zones of myocardium with infarction.

Immunohistochemistry.

Rats were euthanized under isoflurane, their hearts harvested and washedin PBS to remove remnant blood. Hearts were fixed in 4% paraformaldehydeovernight and equilibrated in 15 and 30% sucrose consecutively forcryoprotection. After mounting in OCT (Tissue-Tek), serial sections wereperformed from apex to base at 20 microns (Leica CM1800 Cryostat) andsections were mounted on glass slides. Slides were dried at 37° C. andwashed twice in 1×PBS. Primary antibodies were against Ki67 (clone SP6,1:100; Abcam), CD31, (1:50, BD Biosciences), and smooth muscle alphaactin (1:500, Sigma). Primary antibodies were detected with secondaryantibodies conjugated to Alexa 594 (1:2000). Slides were mounted inVectashield with DAPI (Vector Labs). Sections were imaged byepifluoresence deconvolution microscopy (Leica DM6000B; Leica) withLeica FW4000 software.

Statistical Analysis.

Comparisons of parameters among the three groups were made with one-wayanalysis of variance (ANOVA) followed by Scheffé's multiple comparisontest. Comparisons of parameters between two groups were made by unpairedStudent's t-test. P<0.05 was considered significant.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

This application may be related to U.S. patent application Ser. No.13/220,555, which is a continuation-in-part application of InternationalPatent Application No.: PCT/US2010/001540, filed May 26, 2010, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/181,071,filed May 26, 2009, the disclosures of which are hereby incorporatedherein in their entireties by reference.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1-24. (canceled)
 25. A primed cell, wherein the primed cell comprises acardiac cell or progenitor thereof contacted with a compositioncomprising: (i) one or more of: connective tissue growth factor (CTGF)having at least 95% amino acid sequence identity to SEQ ID NO:1 andhuman C-terminal CTGF peptide having at least 95% amino acid sequenceidentity to SEQ ID NO:2 or SEQ ID NO:3; and (ii) one or more of: insulincomprising polypeptides having at least 95% amino acid sequence identityto any one of SEQ ID NOs:5-7 and IGF-1 having at least 95% amino acidsequence identity to SEQ ID NO:8.
 26. The primed cell of claim 25,wherein the cardiac cell or progenitor thereof is autologous orheterologous.
 27. The primed cell of claim 25, wherein the cardiac cellor progenitor thereof is selected from the group consisting of: an adultcardiac myocyte, an adult cardiac endothelial cell, an adult cardiacsmooth muscle cell, an adult cardiac fibroblast, an adult cardiac stemcell, an adult cardiac progenitor cell, an adult vascular stem cell, anadult epicardial cell, an adult sub-epicardial cell, an adult bonemarrow-derived stem or progenitor cell, a cardiac derivative fromembryonic stem (ES) cells, a cardiac derivative from induced pluripotentstem (iPS) cells, and any combinations thereof.
 28. The primed cell ofclaim 25, wherein the connective tissue growth factor (CTGF) comprisesan amino acid sequence of SEQ ID NO:1 and/or the human C-terminal CTGFpeptide comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.29. The primed cell of claim 25, wherein the insulin polypeptidecomprises an amino acid sequence of any one of SEQ ID NO:5, SEQ ID NO:6,or SEQ ID NO:7 and/or IGF-1 comprises an amino acid sequence of SEQ IDNO:8.
 30. A method, comprising: administering the primed cell of claim25 to a subject in need thereof, wherein the subject suffers fromcardiac tissue damage or a cardiac disease.
 31. The method of claim 30,wherein the method enhances engraftment of the primed cell.
 32. Themethod of claim 31, wherein the cardiac cell or progenitor thereof isselected from the group consisting of: an adult cardiac myocyte, anadult cardiac endothelial cell, an adult cardiac smooth muscle cell, anadult cardiac fibroblast, an adult cardiac stem cell, an adult cardiacprogenitor cell, an adult vascular stem cell, an adult epicardial cell,an adult sub-epicardial cell, an adult bone marrow-derived stem orprogenitor cell, a cardiac derivative from embryonic stem (ES) cells, acardiac derivative from induced pluripotent stem (iPS) cells, and anycombinations thereof.
 33. The method of claim 30, wherein the methodincreases cardiac cell number or reduces cardiac cell death.
 34. Themethod of claim 33, wherein the method increases cardiac cell number byat least 5% compared to a corresponding untreated control cardiac tissueor heart.
 35. The method of claim 30, wherein the method amelioratesischemic damage.
 36. The method of claim 30, wherein the method reducesapoptosis.
 37. The method of claim 30, wherein the method increases cellproliferation.
 38. The method of claim 30, wherein the methodameliorates ischemic damage in a cardiac tissue post-myocardialinfarction.
 39. The method of claim 30, wherein the subject has adisease or a disorder selected from the group consisting of: amyocardial infarction, congestive heart failure, a stroke, and ischemia.40. The method of claim 30, wherein administering occurs directly to asite of cardiac tissue damage or cardiac disease of the subject.
 41. Themethod of claim 30, wherein administering occurs systemically.
 42. Themethod of claim 30, wherein administering occurs by intra-arterialinfusion.
 43. The method of claim 31, wherein engraftment occurs into aheart of the subject in need thereof.
 44. The method of claim 30,wherein the subject in need thereof suffers from a post-myocardialinfarction.